Optoinjection methods

ABSTRACT

Optoinjection method for transiently permeabilizing a target cell by (a) illuminating a population of cells contained in a frame; (b) detecting at least one property of light directed from the frame; (c) locating a target cell by the property of light; and (d) irradiating the target cell with a pulse of radiation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/814,966, filed on Mar. 30, 2004; which is a continuation of U.S.patent application Ser. No. 09/961,691, filed on Sep. 21, 2001, now U.S.Pat. No. 6,753,161; which is a continuation-in-part of U.S. patentapplication Ser. No. 09/728,281, filed Nov. 30, 2000, now U.S. Pat. No.6,514,722; which is a continuation-in-part of U.S. patent applicationSer. No. 09/451,659, filed Nov. 30, 1999, now U.S. Pat. No. 6,534,308;which is a continuation-in-part of U.S. patent application Ser. No.09/049,677, filed Mar. 27, 1998, now U.S. Pat. No. 6,143,535; which is acontinuation-in-part of U.S. patent application Ser. No. 08/824,968,filed Mar. 27, 1997, now U.S. Pat. No. 5,874,266, each of which isincorporated by reference herein.

BACKGROUND OF THE INVENTION

This invention relates to methods for cell manipulation and morespecifically to methods for transiently permeabilizing a cell so that avariety of exogenous materials, such as expressible foreign DNA, can beloaded into the cell.

Previous loading methods have included chemical treatments,microinjection, electroporation and particle bombardment. However, thesetechniques can be time-consuming and suffer from low yields or poor cellsurvival. Another technique termed “optoporation” has used lightdirected toward cells and the surrounding media to induce shock waves,thereby causing small holes to form temporarily in the surface of nearbycells, allowing materials to non-specifically enter cells in the area.Another technique termed “optoinjection” also uses light, but directsthe light to specific cells. Nevertheless, previous light-basedimplementations techniques have suffered from the same disadvantages asother loading techniques.

Thus, there is a need for a method for rapid and efficient loading of avariety of exogenous molecules into cells, with high cell survivalrates. The present invention satisfies this need and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

The present invention provides optoinjection methods for transientlypermeabilizing a target cell. In the general method, the steps are (a)illuminating a population of cells contained in a frame; (b) detectingat least one property of light directed from the frame; (c) locating atarget cell by the property of light; and (d) irradiating the targetcell with a pulse of radiation.

In particular embodiments, a static representation is obtained when thepopulation of cells is substantially stationary; the cells areilluminated through a lens having a numerical aperture of at most 0.5;the pulse of radiation has a diameter of at least 10 microns at thepoint of contact with the target cell; or the resulting pulse ofradiation delivers at most 1 .mu.J/.mu.m.sup.2. As a result, the methodprovides rapid and efficient loading of a variety of exogenous moleculesinto cells, with high cell survival rates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of one embodiment of a cell treatmentapparatus and illustrates the outer design of the housing and display.

FIG. 2 is a perspective view of one embodiment of a cell treatmentapparatus with the outer housing removed and the inner componentsillustrated.

FIG. 3 is a block diagram of the optical subassembly design within oneembodiment of a cell treatment apparatus.

FIG. 4 is a perspective view of one embodiment of an optical subassemblywithin one embodiment of a cell treatment apparatus.

FIG. 5 is a side view of one embodiment of an optical subassembly thatillustrates the arrangement of the scanning lens and the movable stage.

FIG. 6 is a bottom perspective view of one embodiment of an opticalsubassembly.

FIG. 7 is a top perspective view of the movable stage of the celltreatment apparatus.

FIG. 8 shows cells under broad-spectrum light (8A), cells showingloading of Texas-Red-Dextran (8B) and nonviable cells (8C).

FIG. 9 illustrates that the efficiency of optoinjection is energydose-dependent

FIG. 10 compares expression of a plasmid in optoinjected cells (10B)compared to control cells without optoinjection (10A).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A method and apparatus is described for selectively identifying, andindividually targeting with an energy beam, specific cells within a cellpopulation for the purpose of inducing a response in the targeted cells.The population of cells can be a mixed population or homogenous inorigin. The responses of any of the embodiments of the methods andapparatuses of the invention can be lethal or non-lethal. Examples ofsuch responses are set forth above and throughout this disclosure. Thecells targeted can be labeled as is often the case when the specimen isa mixed population. On the other hand, when the specimen is homogenous,the targeted cells can be those individual cells that are beinginterrogated or intersected by the illumination source or the energybeam, in order to study the response of the cell. For instance, suchresponses include the morphological or physiological characteristics ofthe cell. Generally, the method first employs a label that acts as amarker to identify and locate individual cells of a first population ofcells within a cell mixture that is comprised of the first population ofcells and a second population of cells. The cells targeted by theapparatus and methods herein are those that are selectively labeled, inthe case of a mixed population of cells, or the ones undergoinginterrogation or intersection by the illumination source or energy beam.

The chosen label can be any that substantially identifies anddistinguishes the first population of cells from the second populationof cells. For example, monoclonal antibodies that are directly orindirectly tagged with a fluorochrome can be used as specific labels.Other examples of cell surface binding labels include non-antibodyproteins, lectins, carbohydrates, or short peptides with selective cellbinding capacity. Membrane intercalating dyes, such as PKH-2 and PKH-26,could also serve as a useful distinguishing label indicating mitotichistory of a cell. Many membrane-permeable reagents are also availableto distinguish living cells from one another based upon selectedcriteria. For example, phalloidin indicates membrane integrity,tetramethyl rhodamine methyl ester (TMRM) indicates mitochondrialtransmembrane potential, monochlorobimane indicates glutathionereductive stage, carboxymethyl fluorescein diacetate (CMFDA) indicatesthiol activity, carboxyfluorescein diacetate indicates intracellular pH,fura-2 indicates intracellular Ca.sup.2+ level, and5,5′,6,6′-tetrachloro-1,1′1,3,3′-tetra-ethylbenzimidazolo carbocyanineiodide (JC-1) indicates membrane potential. Cell viability can beassessed by the use of fluorescent SYTO 13 or YO PRO reagents.Similarly, a fluorescently-tagged genetic probe (DNA or RNA) could beused to label cells which carry a gene of interest, or express a gene ofinterest. Further, cell cycle status could be assessed through the useof Hoechst 33342 dye to label existing DNA combined withbromodeoxyuridine (BrdU) to label newly synthesized DNA.

It should be noted that if no specific label is available for cells ofthe first population, the method can be implemented in an inversefashion by utilizing a specific label for cells of the secondpopulation. For example, in hematopoietic cell populations, the CD34 orACC-133 cell markers can be used to label only the primitivehematopoietic cells, but not the other cells within the mixture. In thisembodiment, cells of the first population are identified by the absenceof the label, and are thereby targeted by the energy beam.

After cells of the first population are identified, an energy beam, suchas from a laser, collimated or focused non-laser light, RF energy,accelerated particle, focused ultrasonic energy, electron beam, or otherradiation beam, is used to deliver a targeted dose of energy thatinduces the pre-determined response in each of the cells of the firstpopulation, without substantially affecting cells of the secondpopulation.

One such pre-determined response is photobleaching. In photobleaching, alabel in the form of a dye, such as rhodamine 123, GFP, fluoresceinisothiocyanate (FITC), or phycoerythrin, is added to the specimen beforethe instant methods are commenced. After the population of cells hastime to interact with the dye, the energy beam is used to bleach aregion of individual cells in the population. Such photobleachingstudies can be used to study the motility, replenishment, dynamics andthe like of cellular components and processes.

Another response is internal molecular uncaging. In such a process, thespecimen is combined with a caged molecule prior to the commencement ofthe instant methods. Such caged molecules include the.beta.-2,6-dinitrobenzyl ester of L-aspartic acid or the1-(2-nitrophenyl)ethyl ether of 8-hydroxylpyrene-1,3,6-tris-sulfonicacid. Similarly, caging groups including alphacarboxyl-2-nitrobenzyl(CNB) and 5-carboxylmethoxy-2-nitrobenzyl (CMNB) can be linked tobiologically active molecules as ethers, thioethers, esters, amines, orsimilar functional groups. The term “internal molecular uncaging” refersto the fact that the molecular uncaging takes place on the surface orwithin the cell. Such uncaging experiments study rapid molecularprocesses such as cell membrane permeability and cellular signaling.

Yet another response is external molecular uncaging. This usesapproximately the same process as internal molecular caging. However, inexternal molecular uncaging, the uncaged molecule is not attached to orincorporated into the targeted cells. Instead, the responses of thesurrounding targeted cells to the caged and uncaged variants of themolecule are imaged by the instant apparatus and methods.

FIG. 1 is an illustration of one embodiment of a cell treatmentapparatus 10. The cell treatment apparatus 10 includes a housing 15 thatstores the inner components of the apparatus. The housing includes lasersafety interlocks to ensure safety of the user, and also limitsinterference by external influences (e.g., ambient light, dust, etc.).Located on the upper portion of the housing 15 is a display unit 20 fordisplaying captured images of cell populations during treatment. Theseimages are captured by a camera, as will be discussed more specificallybelow. A keyboard 25 and mouse 30 are used to input data and control theapparatus 10. An access door 35 provides access to a movable stage thatholds a specimen container of cells undergoing treatment.

An interior view of the apparatus 10 is provided in FIG. 2. Asillustrated, the apparatus 10 provides an upper tray 200 and lower tray210 that hold the interior components of the apparatus. The upper tray200 includes a pair of intake filters 215A,B that filter ambient airbeing drawn into the interior of the apparatus 10. Below the access door35 is the optical subassembly (not shown). The optical subassembly ismounted to the upper tray 200 and is discussed in detail with regard toFIGS. 3 to 6.

On the lower tray 210 is a computer 225 which stores the softwareprograms, commands and instructions that run the apparatus 10. Inaddition, the computer 225 provides control signals to the treatmentapparatus through electrical signal connections for steering the laserto the appropriate spot on the specimen in order to treat the cells.

As illustrated, a series of power supplies 230A,B,C provide power to thevarious electrical components within the apparatus 10. In addition, anuninterruptable power supply 235 is incorporated to allow the apparatusto continue functioning through short external power interruptions.

FIG. 3 provides a layout of one embodiment of an optical subassemblydesign 300 within an embodiment of a cell treatment apparatus 10. Asillustrated, an illumination laser 305 provides a directed laser outputthat is used to excite a particular label that is attached to targetedcells within the specimen. In this embodiment, the illumination laseremits light at a wavelength of 532 nm. Once the illumination laser hasgenerated a light beam, the light passes into a shutter 310 whichcontrols the pulse length of the laser light.

After the illumination laser light passes through the shutter 310, itenters a ball lens 315 where it is focused into a SMA fiber opticconnector 320. After the illumination laser beam has entered the fiberoptic connector 320, it is transmitted through a fiber optic cable 325to an outlet 330. By passing the illumination beam through the fiberoptic cable 325, the illumination laser 305 can be positioned anywherewithin the treatment apparatus and thus is not limited to only beingpositioned within a direct light pathway to the optical components. Inone embodiment, the fiber optic cable 325 is connected to a vibratingmotor 327 for the purpose of mode scrambling and generating a moreuniform illumination spot.

After the light passes through the outlet 330, it is directed into aseries of condensing lenses in order to focus the beam to the properdiameter for illuminating one frame of cells. As used herein, one frameof cells is defined as the portion of the biological specimen that iscaptured within one frame image captured by the camera. This isdescribed more specifically below.

Accordingly, the illumination laser beam passes through a firstcondenser lens 335. In embodiment, this first lens has a focal length of4.6 mm. The light beam then passes through a second condenser lens 340which, in one embodiment, provides a 100 mm focal length Finally, thelight beam passes into a third condenser lens 345, which preferablyprovides a 200 mm focal length. While the present invention has beendescribed using specific condenser lenses, it should be apparent thatother similar lens configurations that focus the illumination laser beamto an advantageous diameter would function similarly. Thus, thisinvention is not limited to the specific implementation of anyparticular condenser lens system.

Once the illumination laser beam passes through the third condenser lens345, it enters a cube beam splitter 350 that is designed to transmit the532 nm wavelength of light emanating from the illumination laser.Preferably, the cube beam splitter 350 is a 25.4 mm square cube(Melles-Griot, Irvine, Calif.). However, other sizes are anticipated tofunction similarly. In addition, a number of plate beam splitters orpellicle beam splitters could be used in place of the cube beam splitter350 with no appreciable change in function.

Once the illumination laser light has been transmitted through the cubebeam splitter 350, it reaches a long wave pass mirror 355 that reflectsthe 532 nm illumination laser light to a set of galvanometer mirrors 360that steer the illumination laser light under computer control to ascanning lens (Special Optics, Wharton, N.J.) 365, which directs theillumination laser light to the specimen (not shown). The galvanometermirrors are controlled so that the illumination laser light is directedat the proper cell population (i.e. frame of cells) for imaging. The“scanning lens” described in this embodiment of the invention includes arefractive lens. It should be noted that the term “scanning lens” asused in the present invention includes, but is not limited to, a systemof one or more refractive or reflective optical elements used alone orin combination. Further, the “scanning lens” may include a system of oneor more diffractive elements used in combination with one or morerefractive and/or reflective optical elements. One skilled in the artwill know how to design a “scanning lens” system in order to illuminatethe proper cell population.

The light from the illumination laser is of a wavelength that is usefulfor illuminating the specimen. In this embodiment, energy from acontinuous wave 532 nm Nd:YAG frequency-doubled laser (B&W Tek, Newark,Del.) reflects off the long wave pass mirror (Custom Scientific,Phoenix, Ariz.) and excites fluorescent tags in the specimen. In oneembodiment, the fluorescent tag is phycoerythrin. Alternatively, Alexa532 (Molecular Probes, Eugene, Oreg.) can be used. Phycoerythrin andAlexa 532 have emission spectra with peaks near 580 nm, so that theemitted fluorescent light from the specimen is transmitted via the longwave pass mirror to be directed into the camera. The use of the filterin front of the camera blocks light that is not within the wavelengthrange of interest, thereby reducing the amount of background lightentering the camera.

It is generally known that many other devices could be used in thismanner to illuminate the specimen, including, but not limited to, an arclamp (e.g., mercury, xenon, etc.) with or without filters, alight-emitting diode (LED), other types of lasers, etc. Advantages ofthis particular laser include high intensity, relatively efficient useof energy, compact size, and minimal heat generation. It is alsogenerally known that other fluorochromes with different excitation andemission spectra could be used in such an apparatus with the appropriateselection of illumination source, filters, and long and/or short wavepass mirrors. For example, allophycocyanin (APC) could be excited with a633 nm HeNe illumination laser, and fluoroisothiocyanate (FITC) could beexcited with a 488 nm Argon illumination laser. One skilled in the artcould propose many other optical layouts with various components inorder to achieve the objective of this invention.

In addition to the illumination laser 305, an optional treatment laser400 is present to irradiate the targeted cells once they have beenidentified by image analysis. Of course, in one embodiment, thetreatment induces necrosis of targeted cells within the cell population.As shown, the treatment laser 400 outputs an energy beam of 523 nm thatpasses through a shutter 410. Although the exemplary laser outputs anenergy beam having a 523 nm wavelength, other sources that generateenergy at other wavelengths are also within the scope or the presentinvention.

Once the treatment laser energy beam passes through the shutter 410, itenters a beam expander (Special Optics, Wharton, N.J.) 415 which adjuststhe diameter of the energy beam to an appropriate size at the plane ofthe specimen. Following the beam expander 415 is a half-wave plate 420which controls the polarization of the beam. The treatment laser energybeam is then reflected off a mirror 425 and enters the cube beamsplitter 350. The treatment laser energy beam is reflected by 90 degreesin the cube beam splitter 350, such that it is aligned with the exitpathway of the illumination laser light beam. Thus, the treatment laserenergy beam and the illumination laser light beam both exit the cubebeam splitter 350 along the same light path. From the cube beam splitter350, the treatment laser beam reflects off the long wave pass mirror355, is steered by the galvanometers 360, thereafter contacts thescanning lens 365, and finally is focused upon a targeted cell withinthe specimen. Again, the “scanning lens” described in this embodimentincludes a refractive lens. As previously mentioned, the term “scanninglens” includes, but is not limited to, a system of one or morerefractive or reflective optical elements used alone or in combination.Further, the “scanning lens” may include one or more diffractiveelements used in combination with one or more refractive and/orreflective elements. One skilled in the art will know how to design a“scanning lens” system in order to focus upon the targeted cell withinthe specimen.

It should be noted that a small fraction of the illumination laser lightbeam passes through the long wave pass mirror 355 and enters a powermeter sensor (Gentec, Palo Alto, Calif.) 445. The fraction of the beamentering the power sensor 445 is used to calculate the level of poweremanating from the illumination laser 305. In an analogous fashion, asmall fraction of the treatment laser energy beam passes through thecube beam splitter 350 and enters a second power meter sensor (Gentec,Palo Alto, Calif.) 446. The fraction of the beam entering the powersensor 446 is used to calculate the level of power emanating from thetreatment laser 400. The power meter sensors are electrically linked tothe computer system so that instructions/commands within the computersystem capture the power measurement and determine the amount of energythat was emitted.

The energy beam from the treatment laser is of a wavelength that isuseful for achieving a response in the cells. In the example shown, apulsed 523 nm Nd:YLF frequency-doubled laser is used to heat a localizedvolume containing the targeted cell, such that it is induced to diewithin a pre-determined period of time. The mechanism of death isdependent upon the actual temperature achieved in the cell, as reviewedby Niemz, M. H., Laser-tissue interactions: Fundamentals andApplications (Springer-Verlag, Berlin 1996).

A Nd:YLF frequency-doubled, solid-state laser (Spectra-Physics, MountainView, Calif.) is used because of its stability, high repetition rate offiring, and long time of maintenance-free service. However, most cellculture fluids and cells are relatively transparent to light in thisgreen wavelength, and therefore a very high fluence of energy would berequired to achieve cell death. To significantly reduce the amount ofenergy required, and therefore the cost and size of the treatment laser,a dye is purposefully added to the specimen to efficiently absorb theenergy of the treatment laser in the specimen. In the example shown, thenon-toxic dye FD&C red #40 (allura red) is used to absorb the 523 nmenergy from the treatment laser, but one skilled in the art couldidentify other laser/dye combinations that would result in efficientabsorption of energy by the specimen. For example, a 633 nm HeNe laser'senergy would be efficiently absorbed by FD&C green #3 (fast green FCF),a 488 nm Argon laser's energy would be efficiently absorbed by FD&Cyellow #5 (sunset yellow FCF), and a 1064 nm Nd:YAG laser's energy wouldbe efficiently absorbed by Filtron (Gentex, Zeeland, Mich.) infraredabsorbing dye. Through the use of an energy absorbing dye, the amount ofenergy required to kill a targeted cell can be reduced since more of thetreatment laser energy is absorbed in the presence of such a dye.

Another method of achieving thermal killing of cells without theaddition of a dye involves the use of an ultraviolet laser. Energy froma 355 nm Nd:YAG frequency-tripled laser will be absorbed by nucleicacids and proteins within the cell, resulting in thermal heating anddeath. Yet another method of achieving thermal killing of cells withoutthe addition of a dye involves the use of a near-infrared laser. Energyfrom a 2100 nm Ho:YAG laser or a 2940 nm Er:YAG laser will be absorbedby water within the cell, resulting in thermal heating and death.

Although this embodiment describes the killing of cells via thermalheating by the energy beam, one skilled in the art would recognize thatother responses can also be induced in the cells by an energy beam,including photomechanical disruption, photodissociation, photoablation,and photochemical reactions, as reviewed by Niemz (Niemz, supra). Forexample, a photosensitive substance (e.g., hematoporphyrin derivative,tinetiopurpurin, lutetium texaphyrin) (Oleinick and Evans, Thephotobiology of photodynamic therapy: Cellular targets and mechanisms,Rad. Res. 150: S146-S156 (1998)) within the cell mixture could bespecifically activated in targeted cells by irradiation. Additionally, asmall, transient pore could be made in the cell membrane (Palumbo etal., Targeted gene transfer in eukaryotic cells by dye-assisted laseroptoporation, J. Photochem. Photobiol. 36:41-46 (1996)) to allow theentry of genetic or other material. Further, specific molecules in or onthe cell, such as proteins or genetic material, could be inactivated bythe directed energy beam (Grate and Wilson, Laser-mediated,site-specific inactivation of RNA transcripts, PNAS 96:6131-6136 (1999);Jay, D. G., Selective destruction of protein function bychromophore-assisted laser inactivation, PNAS 85:5454-5458 (1988)).Also, photobleaching can be utilized to measure intracellular movementssuch as the diffusion of proteins in membranes and the movements ofmicrotubules during mitosis (Ladha et al., J. Cell Sci., 110(9):1041(1997); Centonze and Borisy, J. Cell Sci. 100 (part 1):205 (1991); Whiteand Stelzer, Trends Cell Biol. 9(2):61-5 (1999); Meyvis, et al., Pharm.Res. 16(8):1153-62 (1999). Further, photolysis or uncaging, includingmultiphoton uncaging, of caged compounds can be utilized to control therelease, with temporal and spacial resolution, of biologically activeproducts or other products of interest (Theriot and Mitchison, J. CellBiol. 119:367 (1992); Denk, PNAS 91(14):6629 (1994)). These mechanismsof inducing a response in a targeted cell via the use of electromagneticradiation directed at specific targeted cells are also intended to beincorporated into the present invention.

In addition to the illumination laser 305 and treatment laser 400, theapparatus includes a camera 450 that captures images (i.e. frames) ofthe cell populations. As illustrated in FIG. 3, the camera 450 isfocused through a lens 455 and filter 460 in order to accurately recordan image of the cells without capturing stray background images. A stop462 is positioned between the filter 460 and mirror 355 in order toeliminate light that may enter the camera from angles not associatedwith the image from the specimen. The filter 460 is chosen to only allowpassage of light within a certain wavelength range. This wavelengthrange includes light that is emitted from the targeted cells uponexcitation by the illumination laser 305, as well as light from aback-light source 475.

The back-light source 475 is located above the specimen to provideback-illumination of the specimen at a wavelength different from thatprovided by the illumination laser 303. This LED generates light at 590nm, such that it can be transmitted through the long wave pass mirror tobe directed into the camera. This back-illumination is useful forimaging cells when there are no fluorescent targets within the framebeing imaged. An example of the utility of this back-light is its use inattaining proper focus of the system, even when there are onlyunstained, non-fluorescent cells in the frame. In one embodiment, theback-light is mounted on the underside of the access door 35 (FIG. 2).

Thus, as discussed above, the only light returned to the camera is fromwavelengths that are of interest in the specimen. Other wavelengths oflight do not pass through the filter 460, and thus do not becomerecorded by the camera 450. This provides a more reliable mechanism forcapturing images of only those cells of interest. It is readily apparentto one skilled in the art that the single filter 460 could be replacedby a movable filter wheel that would allow different filters to be movedin and out of the optical pathway. In such an embodiment, images ofdifferent wavelengths of light could be captured at different timesduring cell processing, allowing the use of multiple cell labels.

It should be noted that in this embodiment, the camera is acharge-coupled device (CCD) and transmits images back to the computersystem for processing. As will be described below, the computer systemdetermines the coordinates of the targeted cells in the specimen byreference to the image captured by the CCD camera.

Referring now to FIG. 4, a perspective view of an embodiment of anoptical subassembly is illustrated. As illustrated, the illuminationlaser 305 sends a light beam through the shutter 310 and ball lens 315to the SMA fiber optic connector 320. The light passes through the fiberoptic cable 325 and through the output 330 into the condenser lenses335, 340 and 345. The light then enters the cube beam splitter 350 andis transmitted to the long wave pass mirror 355. From the long wave passmirror 355, the light beam enters the computer-controlled galvanometers360 and is then steered to the proper frame of cells in the specimenfrom the scanning lens 365.

As also illustrated in the perspective drawing of FIG. 4, the treatmentlaser 400 transmits energy through the shutter 410 and into the beamexpander 415. Energy from the treatment laser 400 passes through thebeam expander 415 and passes through the half-wave plate 420 beforehitting the fold mirror 425, entering the cube beam splitter 350 whereit is reflected 90 degrees to the long wave pass mirror 355, from whichit is reflected into the computer controlled galvanometer mirrors 360.After being steered by the galvanometer mirrors 360 to the scanning lens365, the laser energy beam strikes the proper location within the cellpopulation in order to induce a response in a particular targeted cell.

In order to accommodate a very large surface area of specimen to treat,the apparatus includes a movable stage that mechanically moves thespecimen container with respect to the scanning lens. Thus, once aspecific sub-population (i.e. field) of cells within the scanning lensfield-of-view has been treated, the movable stage brings anothersub-population of cells within the scanning lens field-of-view. Asillustrated in FIG. 5, a computer-controlled movable stage 500 holds aspecimen container (not shown) to be processed. The movable stage 500 ismoved by computer-controlled servo motors along two axes so that thespecimen container can be moved relative to the optical components ofthe instrument. The stage movement along a defined path is coordinatedwith other operations of the apparatus. In addition, specificcoordinates can be saved and recalled to allow return of the movablestage to positions of interest. Encoders on the x and y movement provideclosed-loop feedback control on stage position.

The flat-field (F-theta) scanning lens 365 is mounted below the movablestage. The scanning lens field-of-view comprises the portion of thespecimen that is presently positioned above the scanning lens by themovable stage 500. The lens 365 is mounted to a stepper motor thatallows the lens 365 to be automatically raised and lowered (along thez-axis) for the purpose of focusing the system.

As illustrated in FIGS. 4 to 6, below the scanning lens 365 are thegalvanometer-controlled steering mirrors 360 that deflectelectromagnetic energy along two perpendicular axes. Behind the steeringmirrors is the long wave pass mirror 355 that reflects electromagneticenergy of a wavelength shorter than 545 nm. Wavelengths longer than 545nm are passed through the long wave pass mirror, directed through thefilter 460, coupling lens 455, and into the CCD camera, therebyproducing an image of the appropriate size on the CCD sensor of thecamera 450 (see FIGS. 3 and 4). The magnification defined by thecombination of the scanning lens 365 and coupling lens 455 is chosen toreliably detect single cells while maximizing the area viewed in oneframe by the camera. Although a CCD camera (DVC, Austin, Tex.) isillustrated in this embodiment, the camera can be any type of detectoror image gathering equipment known to those skilled in the art. Theoptical subassembly of the apparatus is preferably mounted on avibration-isolated platform to provide stability during operation asillustrated in FIGS. 2 and 5.

Referring now to FIG. 7, a top view of the movable stage 500 isillustrated. As shown, a specimen container is mounted in the movablestage 500. The specimen container 505 rests on an upper axis nest plate510 that is designed to move in the forward/backward direction withrespect to the movable stage 500. A stepper motor (not shown) isconnected to the upper axis nest plate 510 and computer system so thatcommands from the computer cause forward/backward movement of thespecimen container 505.

The movable stage 500 is also connected to a timing belt 515 thatprovides side-to-side movement of the movable stage 500 along a pair ofbearing tracks 525A,B. The timing belt 515 attaches to a pulley (notshown) housed under a pulley cover 530. The pulley is connected to astepper motor 535 that drives the timing belt 515 to result inside-to-side movement of the movable stage 500. The stepper motor 535 iselectrically connected to the computer system so that commands withinthe computer system result in side-to-side movement of the movable stage500. A travel limit sensor 540 connects to the computer system andcauses an alert if the movable stage travels beyond a predeterminedlateral distance.

A pair of accelerometers 545A,B is preferably incorporated on thisplatform to register any excessive bumps or vibrations that mayinterfere with the apparatus operation. In addition, a two-axisinclinometer 550 is preferably incorporated on the movable stage toensure that the specimen container is level, thereby reducing thepossibility of gravity-induced motion in the specimen container.

The specimen chamber has a fan with ductwork to eliminate condensationon the specimen container, and a thermocouple to determine whether thespecimen chamber is within an acceptable temperature range. Additionalfans are provided to expel the heat generated by the electroniccomponents, and appropriate filters are used on the air intakes 215A,B.

The computer system 225 controls the operation and synchronization ofthe various pieces of electronic hardware described above. The computersystem can be any commercially available computer that can interfacewith the hardware. One example of such a computer system is an IntelPentium II, III or IV-based computer running the Microsoft WINDOWS NToperating system. Software is used to communicate with the variousdevices, and control the operation in the manner that is describedbelow.

When the apparatus is first initialized, the computer loads files fromthe hard drive into RAM for proper initialization of the apparatus. Anumber of built-in tests are automatically performed to ensure theapparatus is operating properly, and calibration routines are executedto calibrate the apparatus. Upon successful completion of theseroutines, the user is prompted to enter information via the keyboard andmouse regarding the procedure that is to be performed. Once the requiredinformation is entered, the user is prompted to open the access door 35and load a specimen onto the movable stage.

Once a specimen is in place on the movable stage and the door is closed,the computer passes a signal to the stage to move into a home position.The fan is initialized to begin warming and defogging of the specimen.During this time, cells within the specimen are allowed to settle to thebottom surface. In addition, during this time, the apparatus may runcommands that ensure that the specimen is properly loaded, and is withinthe focal range of the system optics. For example, specific markings onthe specimen container can be located and focused on by the system toensure that the scanning lens has been properly focused on the bottom ofthe specimen container. Such markings could also be used by theinstrument to identify the container, its contents, and even theprocedure to be performed. After a suitable time, the computer turns offthe fan to prevent excess vibrations during treatment, and cellprocessing begins.

First, the computer instructs the movable stage to be positioned overthe scanning lens so that the first area (i.e. field) of the specimen tobe treated is directly in the scanning lens field-of-view. Thegalvanometer mirrors are instructed to move such that the center framewithin the field-of-view is imaged in the camera. As discussed below,the field imaged by the scanning lens is separated into a plurality offrames. Each frame is the proper size so that the cells within the frameare effectively imaged by the camera.

The back-light 475 is then activated in order to illuminate thefield-of-view so that it can be brought into focus by the scanning lens.Once the scanning lens has been properly focused upon the specimen, thecomputer system divides the field-of-view into a plurality of frames sothat each frame is analyzed separately by the camera. This methodologyallows the apparatus to process a plurality of frames within a largefield-of-view without moving the mechanical stage. Because thegalvanometers can move from one frame to the next very rapidly comparedto the mechanical steps involved in moving the stage, this methodresults is an extremely fast and efficient apparatus.

Other means of ensuring that the specimen is in focus are alsoavailable. For example, a laser proximeter (Cooke Corp., Auburn, Mich.)could rapidly determine the distance between the scanning lens and thesample, and adjust the scanning lens position accordingly. Ultrasonicproximeters are also available, and would achieve the same objective.One skilled in the art could propose other means of ensuring that thespecimen is in focus above the scanning lens. in one preferredembodiment, the apparatus described herein processes at least 1, 2, 3,4, 5, 6, 7, or 14 square centimeters of a biological specimen perminute. In another embodiment, the apparatus described herein processesat least 0.25, 0.5, 1, 2, 3, 4 or 8 million cells of a biologicalspecimen per minute. In one other embodiment, the apparatus canpreferably induce a response in targeted cells at a rate of 50, 100,150, 200, 250, 300, 350, 400 or 800 cells per second.

Initially, an image of the frame at the center of the field-of-view iscaptured by the camera and stored to a memory in the computer.Instructions in the computer analyze the focus of the specimen bylooking at the size of, number of, and other object features in theimage. If necessary, the computer instructs the z-axis motor attached tothe scanning lens to raise or lower in order to achieve the best focus.The apparatus may iteratively analyze the image at several z-positionsuntil the best focus is achieved. The galvanometer-controlled mirrorsare then instructed to image a first frame, within the field-of-view, inthe camera. For example, the entire field-of-view might be divided into4, 9, 12, 18, 24 or more separate frames that will be individuallycaptured by the camera. Once the galvanometer mirrors are pointed to thefirst frame in the field-of-view, the shutter in front of theillumination laser is opened to illuminate the first frame through thegalvanometer mirrors and scanning lens. The camera captures an image ofany fluorescent emission from the specimen in the first frame of cells.Once the image has been acquired, the shutter in front of theillumination laser is closed and a software program (Epic, BuffaloGrove, Ill.) within the computer processes the image.

The power sensor 445 discussed above detects the level of light that wasemitted by the illumination laser, thereby allowing the computer tocalculate if it was adequate to illuminate the frame of cells. If not,another illumination and image capture sequence is performed. Repeatedfailure to sufficiently illuminate the specimen will result in an errorcondition that is communicated to the operator.

Shuttering of illumination light reduces undesirable heating andphotobleaching of the specimen and provides a more repeatablefluorescent signal. An image analysis algorithm is run to locate the x-ycentroid coordinates of all targeted cells in the frame by reference tofeatures in the captured image. If there are targets in the image, thecomputer calculates the two-dimensional coordinates of all targetlocations in relation to the movable stage position and field-of-view,and then positions the galvanometer-controlled mirrors to point to thelocation of the first target in the first frame of cells. It should benoted that only a single frame of cells within the field-of-view hasbeen captured and analyzed at this point. Thus, there should be arelatively small number of identified targets within this sub-populationof the specimen. Moreover, because the camera is pointed to a smallerpopulation of cells, a higher magnification is used so that each targetis imaged by many pixels within the CCD camera.

Once the computer system has positioned the galvanometer controlledmirrors to point to the location of the first targeted cell within thefirst frame of cells, the treatment laser is fired for a brief intervalso that the first targeted cell is given an appropriate dose of energy.The power sensor 446 discussed above detects the level of energy thatwas emitted by the treatment laser, thereby allowing the computer tocalculate if it was adequate to induce a response in the targeted cell.If not sufficient, the treatment laser is fired at the same targetagain. If repeated shots do not deliver the required energy dose, anerror condition is communicated to the operator. These targeting,firing, and sensing steps are repeated by the computer for all targetsidentified in the captured frame.

Once all of the targets have been irradiated with the treatment laser inthe first frame of cells, the mirrors are then positioned to the secondframe of cells in the field-of-view, and the processing repeats at thepoint of frame illumination and camera imaging. This processingcontinues for all frames within the field-of-view above the scanninglens. When all of these frames have been processed, the computerinstructs the movable stage to move to the next field-of-view in thespecimen, and the process repeats at the back-light illumination andauto-focus step. Frames and fields-of-view are appropriately overlappedto reduce the possibility of inadvertently missing areas of thespecimen. Once the specimen has been fully processed, the operator issignaled to remove the specimen, and the apparatus is immediately readyfor the next specimen.

Although the text above describes the analysis of fluorescent images forlocating targets, one can easily imagine that the non-fluorescentback-light LED illumination images will be useful for locating othertypes of targets as well, even if they are unlabeled.

The advantage of using the galvanometer mirrors to control the imagingof successive frames and the irradiation of successive targets issignificant. One brand of galvanometer is the Cambridge Technology, Inc.model number 6860 (Cambridge, Mass.). This galvanometer can repositionvery accurately within a few milliseconds, making the processing oflarge areas and many targets possible within a reasonable amount oftime. In contrast, the movable stage is relatively slow, and istherefore used only to move specified areas of the specimen into thescanning lens field-of-view. Error signals continuously generated by thegalvanometer control boards are monitored by the computer to ensure thatthe mirrors are in position and stable before an image is captured, orbefore a target is fired upon, in a closed-loop fashion.

In the context of the present invention, the term “specimen” has a broadmeaning. It is intended to encompass any type of biological sampleplaced within the apparatus. The specimen may be enclosed by, orassociated with, a container to maintain the sterility and viability ofthe cells. Further, the specimen may incorporate, or be associated with,a cooling apparatus to keep it above or below ambient temperature duringoperation of the methods described herein. The specimen container, ifone is used, must be compatible with the use of the illumination laser,back-light illuminator, and treatment laser, such that it transmitsadequate energy without being substantially damaged itself.

Of course, many variations of the above-described embodiment arepossible, including alternative methods for illuminating, imaging, andtargeting the cells. For example, movement of the specimen relative tothe scanning lens could be achieved by keeping the specimensubstantially stationary while the scanning lens is moved. Steering ofthe illumination beam, images, and energy beam could be achieved throughany controllable reflective or diffractive device, including prisms,piezo-electric tilt platforms, or acousto-optic deflectors.Additionally, the apparatus can image/process from either below or abovethe specimen. Because the apparatus is focused through a movablescanning lens, the illumination and energy beams can be directed todifferent focal planes along the z-axis. Thus, portions of the specimenthat are located at different vertical heights can be specificallyimaged and processed by the apparatus in a three-dimensional manner. Thesequence of the steps could also be altered without changing theprocess. For example, one might locate and store the coordinates of alltargets in the specimen, and then return to the targets to irradiatethem with energy one or more times over a period of time.

To optimally process the specimen, it should be placed on asubstantially flat surface so that a large portion of the specimenappears within a narrow range of focus, thereby reducing the need forrepeated auto-focus steps. The density of cells on this surface can, inprinciple, be at any value. However, the cell density should be as highas possible to minimize the total surface area required for theprocedure.

A further embodiment of the invention provides optoinjection methods fortransiently permeabilizing a target cell. In the general method, thesteps are (a) illuminating a population of cells contained in a frame;(b) detecting at least one property of light directed from the frame;(c) locating a target cell by the property of light; and (d) irradiatingthe target cell with a pulse of radiation.

The “cells” used in the method can be any, biological cells, includingprocaryotic and eucaryotic cells, such as animal cells, plant cells,yeast cells, human cells and non-human primate cells. The cells can betaken from organisms or harvested from cell cultures. The method canalso be applied to permeabilize subcellular organelles.

It follows that the term “population” of cells means a group of morethan one of such cells. While performing the method, the population ofcells can be presented in a specimen container such as 505.

The cells can also be associated with an exogenous label such as afluorophore. Other labels useful in the invention have been described indetail above.

The population of cells can be “illuminated” by any source that canprovide light energy, including a laser and an arc lamp. The lightenergy can be of any wavelength, such as visible, ultraviolet andinfrared light. When the light is from a laser, such as 400, usefulwavelengths can range from 100 nm to 1000 nm, 200 nm to 800 nm, 320 nmto 695 nm, and 330 nm to 605 nm. Particular wavelengths include 349 nm,355 nm, 488 nm, 523 nm, 532 nm, 580 nm, 590 nm, 633 nm, 1064 nm, 2100 nmand 2940 nm. Other illumination sources include any source for an energybeam, as described in detail above. The light can then be directed byany conventional means, such as mirrors, lenses and beam-splitters, tothe population of cells.

Once the cells are illuminated, they can be observed in a “frame.” Aspreviously defined, one “frame” of cells is the portion of thebiological specimen that is captured within one frame image captured bythe camera. A particularly useful frame can have an area of at least 50,70, 85, 95 or 115 mm². A useful magnification range for the camera isbetween 2× and 40× and more particularly between 2.5× and 25× and stillmore particularly between 5× and 10×.

When the frame is illuminated, one or more properties of light can thenbe detected from the frame. The detectable properties include lighthaving visible, ultraviolet and infrared wavelengths, the intensity oftransmittance and reflectance, fluorescence, linear and circularpolarization, and phase-contrast illumination. These properties can bedetected by conventional optical devices such as the devices alreadydescribed in detail above.

The target cell can then be located based on its size, shape and otherpreselected visual properties, and then irradiated with a pulse ofradiation. The radiation then causes a temporary permeabilization of thesurface of the target cell. While not limiting the method to aparticular mechanism, it is believed that the light causes localizedmelting or other disruption of the cell membrane's continuity, allowingsmall pores to form without killing the cell.

As a result of transiently permeabilizing the cells, exogenous moleculesin the presence of the cell can then enter the cell, whether bydiffusion or other mechanism. The term “presence of the cell” herein asapplied to an exogenous molecule means in the area near the cell, suchas the surrounding medium, so that if the cell were permeabilized, theexogenous molecule could then enter the cell.

The term “exogenous molecule” herein means any molecule or material thatdoes not naturally occur in the cells of the population. It alsoincludes molecules or materials that may occur naturally in the cell,but in significantly higher concentrations than occur naturally in thecell. Exogenous molecules include nucleic acids, polypeptides,carbohydrates, lipids and small molecules. Particular nucleic acidsinclude RNAs, expression plasmids, expression cassettes and otherexpressible DNA. Particular polypeptides include antibodies and otherproteins, which can be introduced into cells to explore interactionsbetween exogenous and endogenous proteins for applications inproteomics. Other polypeptides include peptides for introduction intoantigen-displaying dendritic cells. Particular carbohydrates includenon-naturally occurring metabolites, such as isotopically labeledsugars, and polysaccharides, such as labeled dextrans. Particular lipidsinclude preselected lipids for incorporation into the cell membrane orother organelles, as well as liposomes and liposomes containing otherexogenous molecules of interest. Particular small molecules includeligands for endogenous receptors to study ligand-receptor binding.Similarly, drugs can be introduced into cells, which, in turn, can beintroduced as a delivery device into a patient for therapeutic purposes.The term also encompasses dyes capable of absorbing visible, ultravioletor infrared light.

Exogenous molecules can have a size of greater than 0.1, 0.2, 0.3, 0.5,1, 2, 3, 5, 10, 20, 30, 50, 70, 100 or even 200 kiloDaltons. Althoughthe efficiency rate of cells that are loaded with at least one exogenousmolecule will vary depending on the size and nature of the exogenousmolecule, loading efficiencies can be as high as 5%, 10%, 20%, 50%, 75%or even 90% of the population of cells. It should also be emphasizedthat the method encompasses techniques where two or more exogenousmolecules are loaded into cells simultaneously or sequentially.

Significantly, as result of using the method, greater than 50%, 60%,70%, 80%, 90%, 95% or even 98% of the irradiated target cells can beviable after completion of the method. Methods for measuring cellsurvival rates are well known in the art and membrane-permeable reagentsfor distinguishing living cells have been described above. For example,preselected reagents can be added to the media before, during or afterperforming the method. Specific examples of useful reagents includeCalcein AM as an indicator of viability and Sytox Blue as an indicatorfor dead cells. Other well-known methods include trypan blue exclusion,propidium iodide and ⁵¹Cr-release assay.

It should be noted that the general method presented above has severalalternate embodiments that are particularly useful.

First, the general method can be used when the population of cells issubstantially stationary. The term “substantially stationary” hereinmeans that the cells are relatively immobile with respect to the mediumand are not in flowing medium, and the cells are not subjected to grossmovement of a container; but, they can be subject to vibrations andslight movements that normally occur in a typical laboratory. While theterm encompasses cells that are immobilized to a surface or within themedium, substantially stationary cells need not be immobilized orotherwise bound to a surface to be considered substantially stationary.Thus; the term includes cells that have settled to the bottom of aspecimen container.

When a population of cells is substantially stationary, it becomesuseful to obtain a static representation of the cells in the frame. Theterm “static representation” herein means a substantially complete imageof the cells taken during a fixed and discrete time period, rather thanas a continuous image, as in a “live” monitor.

Because the cells are substantially stationary, the staticrepresentation can then be used as a reliable indicator of the locationof one or more cells at subsequent points in time. Moreover, a staticrepresentation can be obtained under one set of conditions and anotherstatic representation obtained under a different set of conditions sothat the two representations can be compared usefully without undueconcern for movement of the cells. For example, an image of the cellsunder visible light can be compared with a corresponding fluorescenceimage to identify fluorescently tagged cells of interest among a generalpopulation of cells. The static representation can also be used as thebasis for computer-aided identification and determination of thelocation of a target cell of interest, based on any of the lightproperties discussed above.

Second, the general method can be performed where the population ofcells is illuminated through a lens having numerical aperture of at most0.5, 0.4 or 0.3. The term “numerical aperture” or “N.A.” used herein isdefined N.A.=n(sin μ), where n is the refractive index of the imagingmedium between the lens and the cells, and μ is one-half of the angularaperture.

As a consequence of using a lens having such a low numerical aperture,the lens can have a greater working distance, such as at least 5, 7 or10 mm. The term “working distance” herein means the distance between thefront of the lens to the object, meaning the nearest surface of thepopulation of cells. A particularly useful lens is a flat-field(F-theta) lens, as exemplified by lens 365, described above. It shouldbe noted that confocal microscopy is not possible under such lensparameters.

Third, the pulse of radiation can have a diameter of at least 2, 5, 7,10, 15, 20, 25 or 30 microns at the point of contact with the targetcell. In most cases, the breadth of the radiation will be much widerthan any individual cell. Consequently, the beam of radiation need notbe separately targeted to a particular point on a cell or cell surfaceto be effective, but can be directed to the general area of a cellpopulation without losing effectiveness. As a result, sensitivity tobeam steering accuracy is reduced and throughput is dramaticallyincreased.

Fourth, the energy delivered by the pulse of radiation can be limited toat most 2, 1.5, 1, 0.7, 0.5, 0.3, 0.2, 0.1, 0.05, 0.02, 0.01 or even0.005 μJ/μm². This has the advantage of increasing the survival ratewhile maintaining efficient loading rates. Moreover, unlike previousmethods, the effective energy levels are low enough to allow the use ofcommon plastic specimen containers without damaging the container.

The general method can also be modified to increase throughput. At themost basic level, the direction of the pulse of radiation can beadjusted to irradiate a second target cell in the population in a givenframe. Similarly, subsequent fields of view of the population of cellscan be processed as described above. This is especially useful when thepopulation of cells remains in a substantially stationary locationrelative to the lens. Alternatively, the cells can be moved relative tothe lens between applications of the method for further steps ofdetecting, locating and irradiating cells.

To maximize throughput of the cells, one or more of the steps of themethod can be automated, as exemplified by the apparatus described indetail above. For example, each of the steps can be controlled by amicroprocessor. Similarly, a static representation can be processed asan image or a data set stored in computer memory. By automating each ofthe steps, the optoinjection method can irradiate at least 5,000,10,000, 20,000, 50,000, 70,000, 100,000 or even 150,000 cells per minute

The following examples illustrate the use of the described method andapparatus in different applications.

Example 1 Autologous HSC Transplantation

A patient with a B cell-derived metastatic tumor in need of anautologous HSC transplant is identified by a physician. As a first stepin the treatment, the patient undergoes a standard HSC harvestprocedure, resulting in collection of approximately 1.times.10.sup.10hematopoietic cells with an unknown number of contaminating tumor cells.The harvested cells are enriched for HSC by a commercial immunoaffinitycolumn (ISOLEX 300, Nexell Therapeutics, Irvine, Calif.) that selectsfor cells bearing the CD34 surface antigen, resulting in a population ofapproximately 3.times.10.sup.8 hematopoietic cells, with an unknownnumber of tumor cells. The mixed population is thereafter contacted withanti-B cell antibodies (directed against CD20 and CD22) that areconjugated to phycoerythrin. The labeled antibodies specifically bind tothe B cell-derived tumor cells.

The mixed cell population is then placed in a sterile specimen containeron a substantially flat surface near confluence, at approximately500,000 cells per square centimeter. The specimen is placed on themovable stage of the apparatus described above, and all detectable tumorcells are identified by reference to phycoerythrin and targeted with alethal dose of energy from a treatment laser. The design of theapparatus allows the processing of a clinical-scale transplant specimenin under 4 hours. The cells are recovered from the specimen container,washed, and then cryopreserved. Before the cells are reinfused, thepatient is given high-dose chemotherapy to destroy the tumor cells inthe patient's body. Following this treatment, the processed cells arethawed at 37° C. and are given to the patient intravenously. The patientsubsequently recovers with no remission of the original cancer.

Example 2 Allogeneic HSC Transplantation

In another embodiment, the significant risk and severity ofgraft-versus-host disease in the allogeneic HSC transplant setting canbe combated. A patient is selected for an allogeneic transplant once asuitable donor is found. Cells are harvested from the selected donor asdescribed in the above example. In this case, the cell mixture iscontacted with phycoerythrin-labeled anti-CD3 T-cell antibodies.Alternatively, specific allo-reactive T-cell subsets could be labeledusing an activated T-cell marker (e.g. CD69) in the presence ofallo-antigen. The cell population is processed by the apparatusdescribed herein, thereby precisely defining and controlling the numberof T-cells given to the patient. This type of control is advantageous,because administration of too many T-cells increases the risk ofgraft-versus-host disease, whereas too few T-cells increases the risk ofgraft failure and the risk of losing of the known beneficialgraft-versus-leukemia effect. The present invention and methods arecapable of precisely controlling the number of T-cells in an allogeneictransplant.

Example 3 Tissue Engineering

In another application, the present apparatus is used to removecontaminating cells in inocula for tissue engineering applications. Cellcontamination problems exist in the establishment of primary cellcultures required for implementation of tissue engineering applications,as described by Langer and Vacanti, Tissue engineering: The challengesahead, Sci. Am. 280:86-89 (1999). In particular, chondrocyte therapiesfor cartilage defects are hampered by impurities in the cell populationsderived from cartilage biopsies. Accordingly, the present invention isused to specifically remove these types of cells from the inocula.

For example, a cartilage biopsy is taken from a patient in need ofcartilage replacement. The specimen is then grown under conventionalconditions (Brittberg et al., Treatment of deep cartilage defects in theknee with autologous chondrocyte transplantation, N.E. J. Med.331:889-895 (1994)). The culture is then stained with a specific labelfor any contaminating cells, such as fast-growing fibroblasts. The cellmixture is then placed within the apparatus described and the labeled,contaminating cells are targeted by the treatment laser, therebyallowing the slower growing chondrocytes to fully develop in culture.

Example 4 Stem Cell Therapy

Yet another embodiment involves the use of embryonic stem cells to treata wide variety of diseases. Since embryonic stem cells areundifferentiated, they can be used to generate many types of tissue thatwould find use in transplantation, such as cardiomyocytes and neurons.However, undifferentiated embryonic stem cells that are implanted canalso lead to a jumble of cell types which form a type of tumor known asa teratoma (Pedersen, R. A., Embryonic stem cells for medicine, Sci.Amer. 280:68-73 (1999)). Therefore, therapeutic use of tissues derivedfrom embryonic stem cells must include rigorous purification of cells toensure that only sufficiently differentiated cells are implanted. Theapparatus described herein is used to eliminate undifferentiated stemcells prior to implantation of embryonic stem cell-derived tissue in thepatient.

Example 5 Generation of Human Tumor Cell Cultures

In another embodiment, a tumor biopsy is removed from a cancer patientfor the purpose of initiating a culture of human tumor cells. However,the in vitro establishment of primary human tumor cell cultures frommany tumor types is complicated by the presence of contaminating primarycell populations that have superior in vitro growth characteristics overtumor cells. For example, contaminating fibroblasts represent a majorchallenge in establishing many cancer cell cultures. The disclosedapparatus is used to particularly label and destroy the contaminatingcells, while leaving the biopsied tumor cells intact. Accordingly, themore aggressive primary cells will not overtake and destroy the cancercell line.

Example 6 Generation of a Specific mRNA Expression Library

The specific expression pattern of genes within different cellpopulations is of great interest to many researchers, and many studieshave been performed to isolate and create libraries of expressed genesfor different cell types. For example, knowing which genes are expressedin tumor cells versus normal cells is of great potential value (Cossman,et al., Reed-Stemberg cell genome expression supports a B-cell lineage,Blood 94:411-416 (1999)). Due to the amplification methods used togenerate such libraries (e.g. PCR), even a small number of contaminatingcells will result in an inaccurate expression library (Cossman et al.,supra; Schutze and Lahr, Identification of expressed genes bylaser-mediated manipulation of single cells, Nature Biotechnol.16:737-742 (1998)). One approach to overcome this problem is the use oflaser capture microdissection (LCM), in which a single cell is used toprovide the starting genetic material for amplification (Schutze andLahr, supra). Unfortunately, gene expression in single cells is somewhatstochastic, and may be biased by the specific state of that individualcell at the time of analysis (Cossman et al., supra). Therefore,accurate purification of a significant cell number prior to extractionof mRNA would enable the generation of a highly accurate expressionlibrary, one that is representative of the cell population beingstudied, without biases due to single cell expression or expression bycontaminating cells. The methods and apparatus described in thisinvention can be used to purify cell populations so that nocontaminating cells are present during an RNA extraction procedure.

Example 7 Transfection of a Specific Cell Population

Many research and clinical gene therapy applications are hampered by theinability to transfect an adequate number of a desired cell type withouttransfecting other cells that are present. The method of the presentinvention would allow selective targeting of cells to be transfectedwithin a mixture of cells. By generating a photomechanical shock wave ator near a cell membrane with a targeted energy source, a transient porecan be formed, through which genetic (or other) material can enter thecell. This method of gene transfer has been called optoporation (Palumboet al. supra). The apparatus described above can achieve selectiveoptoporation on only the cells of interest in a rapid, automated,targeted manner.

For example, white blood cells are plated in a specimen container havinga solution containing DNA to be transfected. Fluorescently-labeledantibodies having specificity for stem cells are added into the mediumand bind to the stem cells. The specimen container is placed within thecell processing apparatus and a treatment laser is targeted to any cellsthat become fluorescent under the illumination laser light. Thetreatment laser facilitates transfection of DNA specifically into thetargeted cells.

Example 8 Selection of Desirable Clones in a Biotechnology Application

In many biotechnology processes where cell lines are used to generate avaluable product, it is desirable to derive clones that are veryefficient in producing the product. This selection of clones is oftencarried out manually, by inspecting a large number of clones that havebeen isolated in some manner. The present invention would allow rapid,automated inspection and selection of desirable clones for production ofa particular product. For example, hybridoma cells that are producingthe greatest amounts of antibody can be identified by a fluorescentlabel directed against the Fc region. Cells with no or dim fluorescentlabeling are targeted by the treatment laser for killing, leaving behindthe best producing clones for use in antibody production.

Example 9 Automated Monitoring of Cellular Responses

Automated monitoring of cellular responses to specific stimuli is ofgreat interest in high-throughput drug screening. Often, a cellpopulation in one well of a well-plate is exposed to a stimulus, and afluorescent signal is then captured over time from the cell populationas a whole. Using the methods and apparatus described herein, moredetailed monitoring could be done at the single cell level. For example,a cell population can be labeled to identify a characteristic of asubpopulation of cells that are of interest. This label is then excitedby the illumination laser to identify those cells. Thereafter, thetreatment laser is targeted at the individual cells identified by thefirst label, for the purpose of exciting a second label, therebyproviding information about each cell's response. Since the cells aresubstantially stationary on a surface, each cell could be evaluatedmultiple times, thereby providing temporal information about thekinetics of each cell's response. Also, through the use of the largearea scanning lens and galvanometer mirrors, a relatively large numberof wells could be quickly monitored over a short period of time.

As a specific example, consider the case of alloreactive T-cells aspresented in Example 2, above. In the presence of allo-antigen,activated donor T-cells could be identified by CD69. Instead of usingthe treatment laser to target and kill these cells, the treatment lasercould be used to examine the intracellular pH of every activated T-cellthrough the excitation and emitted fluorescence of carboxyfluoresceindiacetate. The targeted laser allows the examination of only cells thatare activated, whereas most screening methods evaluate the response ofan entire cell population. If a series of such wells are being monitoredin parallel, various agents could be added to individual wells, and thespecific activated T-cell response to each agent could be monitored overtime. Such an apparatus would provide a high-throughput screening methodfor agents that ameliorate the alloreactive T-cell response ingraft-versus-host disease. Based on this example, one skilled in the artcould imagine many other examples in which a cellular response to astimulus is monitored on an individual cell basis, focusing only oncells of interest identified by the first label.

Example 10 Photobleaching Studies

Photobleaching, and/or photobleach recovery, of a specific area of afluorescently-stained biological sample is a common method that is usedto assess various biological processes. For example, a cell suspensionis labeled with rhodamine 123, which fluorescently stains mitochondriawithin the cells. Using the instant illumination laser, the mitochondriawithin one or more cells are visualized due to rhodamine 123fluorescence. The treatment laser is then used to deliver a focused beamof light that results in photobleaching of the rhodamine 123 in a smallarea within one or more cells. The photobleached area(s) then appeardark immediately thereafter, whereas adjacent areas are unaffected. Aseries of images are then taken using the illumination laser, providinga time-lapse series of images that document the migration of unbleachedmitochondria into the area that was photobleached with the treatmentlaser. This approach can be used to assess the motion, turnover, orreplenishment of many biological structures within cells.

Thus, in cultured rat neurites, the photobleach recovery of mitochondriais a measure of the size of the mobile pool of mitochondria within eachcell (Chute, et al., Analysis of the steady-state dynamics organellemotion in cultured neurites, Clin. Exp. Pharmco. Physiol. 22:360(1995)). The rate of photobleach recovery in these cells is dependent onintracellular calcium and magnesium concentrations, energy status, andmicrotubule integrity. Neurotoxic substances, such as taxol orvinblastine, will affect the rate of photobleach recovery. Therefore, anassay for neurotoxic substances could be based on the measurement ofphotobleach recovery of mitochondria within a statistically significantnumber of neurites that had been exposed to various agents in the wellsof a multi-well plate. In such an application, the apparatus describedherein and used as described above, would provide a rapid automatedmethod to assess neurotoxicity of many substances on a large number ofcells. Based on this example, one skilled in the art could imagine manyother examples in which photobleaching is induced and photobleachrecovery is monitored in order to obtain useful information from abiological specimen.

Example 11 Uncaging Studies

Use of caged compounds to study rapid biological processes involves thebinding (i.e. caging) of a biologically relevant substance in aninactive state, allowing the caged substance to diffuse into thebiological specimen (a relatively slow process), and then using a laserto induce a photolysis reaction (a relatively fast process) whichliberates (i.e. uncages) the substance in situ over microsecond timescales. The biological specimen is then observed in short time-lapsemicroscopy in order to determine the effect of the uncaged substance onsome biological process. Cages for many important substances have beendescribed, including Dioxygen, cyclic ADP ribose (cADPR), nicotinic acidadenine dinucleotide phosphate (NAADP), nitric oxide (NO), calcium,L-aspartate, and adenosine triphosphate (ATP). Chemotaxis is one exampleof a physiological characteristic that can be studied by uncagingcompounds.

Uncaging studies involve the irradiation of a portion of a biologicalspecimen with laser light followed by examination of the specimen withtime-lapse microscopy. The apparatus of the current invention has clearutility in such studies. As a specific example, consider the study of E.coli chemotaxis towards L-aspartate (Jasuja et al., Chemotaceticresponses of Escherichia coli to small jumps of photoreleasedL-aspartate, Biophys. J. 76:1706 (1999)). The beta-2,6-dinitrobenzylester of L-aspartic acid and the 1-(2-nitrophenyl)ethyl ether of8-hydroxylpyrene-1,3,6-tris-sulfonic acid are added to the wells of awell plate containing E. coli. Upon irradiation with the treatmentlaser, a localized uncaging of L-aspartate and the fluorophore8-hydroxylpyrene-1,3,6-tris-sulfonic acid (pyranine) is induced. TheL-aspartate acts as a chemoattractant for E. coli., and in subsequentfluorescent images (using the illumination laser) the pyraninefluorophore acts as an indicator of the degree of uncaging that hasoccurred in the local area of irradiation. Time-lapse images of the E.coli. in the vicinity illuminated by visible wavelength light, such asfrom the back-light, of the uncaging event are used to measure thechemotacetic response of the microorganisms to the locally uncagedL-aspartate. Due to the nature of the present invention, a large numberof wells, each with a potential anti-microbial agent added, are screenedin rapid order to determine the chemotacetic response of microorganisms.Based on this example, one skilled in the art could imagine many otherexamples in which uncaging is induced by the treatment laser, followedby time-lapse microscopy in order to obtain useful information on alarge number of samples in an automated fashion.

Example 12 Optoinjection of NIH-3T3 Cells with 70 kD Dextran

This example illustrates an optoinjection method for transientlypermeabilizing a target cell. NIH-3T3 cells were grown in a 96-wellplate. The growth medium was removed and replaced with PBS containing 1%BSA and 0.1 mM Texas-Red-Dextran (70 kDa) (Molecular Probes, Eugene,Oreg.). Upon illumination of the cells under broad-spectrum light, astatic image (FIG. 8A) was obtained to determine which cells to target.

A 30 micron energy beam having a wavelength of 523 nm was directedsequentially to the target cells through a flat-field lens having amagnification of 2.5.times., a numerical aperture (N.A.) of 0.25, and aworking distance of greater than 10 mm. Over 500 cells were targeted persecond.

After irradiating the target cells, the wells were washed, and SytoxBlue (10 mM, Molecular Probes) was added to stain non-viable cells. Asshown in FIG. 8B, about 70% of the cells showed loading of theTexas-Red-Dextran as an exogenous molecule. Moreover, only one cell wasnon-viable (FIG. 8C), equivalent to about a 95% survival rate.

Example 13 Optoinjection of SU-DHL-4 Cells with Sytox Green

Using the same hardware apparatus as in Example 12, SU-DHL-4 cells wereplaced in 96-well plates in PBS with 1% HSA. The membrane-impermeabledye Sytox Green (Molecular Probes) was added at 0.05 mM, the cells wereallowed to settle, and then were imaged and targeted with a 30 micronlaser beam. Different energy levels ranging from 2 to 15 .mu.J per pulseof the laser were used in each of five wells, with each target cellreceiving one pulse. As show in FIG. 9, the efficiency of optoinjectionwas energy dose-dependent, ranging from 58% at 4 mu.J/cell (0.0057.mu.J/.mu.m.sup.2) to 92% at 15 .mu.J/cell (0.021 .mu.J/.mu.m.sup.2). Inall cases, cell viability was greater than 95%.

Example 14 Optoinjection of 293T Cells with pEGFP-N1 Plasmid

In this experiment, the exogenous molecule was a DNA plasmid of 4.3 kbencoding the fluorescent EGFP protein (pEGFP-N1). The same hardwareapparatus was used as in Example 12. The cells were in a medium of PBSand 1% HSA, and then 0.1 microgram of plasmid was added to each well.The cells were imaged, located and targeted with the laser beam suchthat each cell received 1 to 8 pulses of 15 .mu.J each (0.021.mu.J/.mu.m.sup.2). The cells were washed, placed in growth medium, andthen cultured for 48 to 96 hours. After culturing, cells were evaluatedfor the expression of the fluorescent EGFP protein. As shown in FIG. 10,a number of cells displayed the fluorescent phenotype in the treatedwells (FIG. 10A), whereas no fluorescence was observed in the controlwell (FIG. 10B), which were treated identically with the exception ofdelivering the laser pulses.

Although aspects of the present invention have been described byparticular embodiments exemplified herein, the present invention is notso limited. The present invention is only limited by the claims appendedbelow.

1. A method of rapidly introducing an exogenous molecule into a livepopulation of cells, comprising the steps of: providing a livepopulation of cells in the presence of said exogenous molecule;providing an energy source configured to deliver a beam of radiation;positioning a container holding said live population of cells within thefocus of said beam of radiation; and contacting one or more cells ofsaid live population of cells with said beam of radiation, wherein saidbeam of radiation has a diameter of at least 2 μm at the point ofcontact with said one or more cells; wherein one or more cells of saidlive population of cells is transiently permeabilized to allow saidexogenous molecule to enter.
 2. The method of claim 1, wherein saiddiameter of said beam of radiation is at least 5 μm at said point ofcontact with said live population of cells.
 3. The method of claims 1,wherein said beam of radiation delivers at most 2, 1.5, 1, 0.7, 0.5,0.3, 0.2, 0.1, 0.05, 0.02, 0.01 or 0.005 μJ/μm².
 4. The method of claims1, wherein said beam of radiation is delivered through a lens with anumerical aperture of at most 0.5, 0.4, 0.3, or 0.25.
 5. A method ofrapidly introducing exogenous molecules into a live population of cells,comprising the steps of: providing a live population of cells in thepresence of exogenous molecules; providing an energy source configuredto deliver a beam of radiation; positioning a container holding saidlive population of cells within the focus of said beam of radiation; andcontacting one or more cells of said live population of cells with saidbeam of radiation, wherein said beam of radiation delivers at most 2μJ/μm²; wherein one or more cells of said live population of cells istransiently permeabilized to allow said exogenous molecules to enter. 6.The method of claim 5, wherein said beam of radiation delivers a valueselected from the group of at most 1.5, 1, 0.7, 0.5, 0.3, 0.2, 0.1,0.05, 0.02, 0.01 or 0.005 μJ/μm².
 7. The method of claims 5, whereinsaid beam of radiation is delivered through a lens with a numericalaperture of at most 0.5, 0.4, 0.3, or 0.25.
 8. The method of claims 6,wherein said lens has an F-theta flat-field correction.
 9. A method ofrapidly introducing exogenous molecules into a live population of cells,comprising the steps of: providing a live population of cells in thepresence of exogenous molecules; providing an energy source configuredto deliver a beam of radiation; positioning a container holding saidlive population of cells within the focus of said beam of radiation; andcontacting one or more cells of said live population of cells with saidbeam of radiation, wherein said beam of radiation is delivered through alens with a numerical aperture of at most 0.5; wherein one or more cellsof said live population of cells is transiently permeabilized to allowsaid exogenous molecules to enter.
 10. The method of claim 9, whereinsaid lens has a numerical aperture selected from the group of at most0.4, 0.3, or 0.25.
 11. The method of claims 9, wherein positioning saidcontainer holding said live population of cells within said focus ofsaid beam of radiation further comprises the steps of: illuminating afield-of-view containing said live population of cells; analyzing animage of said live population of cells within said field-of-view; andadjusting the distance between said population of cells and said lens.12. The method of claims 9, wherein contacting said live population ofcells with said beam of radiation comprises: pulsing said energy source;and steering the pulsed beam of radiation from said energy source tomultiple spots within said live population of cells.
 13. The method ofclaim 11, wherein said pulsed beam of radiation is wider than anyindividual cell and is directed to the general area of said livepopulation of cells.
 14. The method of claims 9, wherein said lenscomprises a working distance of at least 5, 7, or 10 mm.
 15. A method ofrapidly introducing an exogenous molecule into a live cell, comprising:providing a live cell in the presence of said exogenous molecule;providing an energy source configured to deliver a beam of radiation;and contacting said live cell with said beam of radiation, wherein saidlive cell is transiently permeabilized to allow said exogenous moleculeto enter; and wherein (a) said beam of radiation has a diameter of atleast 2 μm at the point of contact with said live cell, (b) said beam ofradiation delivers at most 2 μJ/μm², (c) said beam of radiation isdelivered through a lens with a numerical aperture of at most 0.5, or(d) two or more of (a), (b) or (c).
 16. The method of claim 16, whereinsaid beam of radiation has a diameter of at least 2, 5, 7, 10, 15, 20,25, or 30 μm at the point of contact with said live cell.
 17. The methodof claim 15, wherein said beam of radiation is wider than the cell or isdirected to the general area of said cell.
 18. The method of claim 15,wherein two or more of the following apply (a) said beam of radiationhas a diameter of at least 2 μm at the point of contact with said livecell, (b) said beam of radiation delivers at most 2 μJ/μm², or (c) saidbeam of radiation is delivered through a lens with a numerical apertureof at most 0.5.
 19. The method of claim 18, wherein said beam ofradiation has a diameter of at least 10 μm at the point of contact withsaid live cell.
 20. The method of claim 18, wherein (a), (b) and (c)apply.